The Na-K-Cl cotransporter (NKCC) is a plasma membrane transport protein that plays a central role in cellular homeostasis. In non-polarized cells including neurons the NKCC1 isoform is involved in regulation of intracellular chloride and cell volume, and in secretory epithelia, NKCC1 functions together with Cl channels, the Na pump, and K channels to bring about regulated salt movement. In the mammalian kidney another isoform, NKCC2, mediates salt absorption and is the site of action of the loop diuretic drugs furosemide and bumetanide. NKCCs are members of the cation-chloride cotransporter family, and of the APC superfamily of transporters. The long term goal of this project, which focuses on NKCC1, is to understand the molecular mechanism of the cotransporter, including the structural and functional features underlying ion translocation and its regulation, and the significance of the transporter in cell and organ function. The research is directly relevant to the understanding, diagnosis, and treatment of ion transport diseases and disease conditions including hypertension, polycystic kidney disease, secretory diarrhea, cerebral edema associated with stroke, and cystic fibrosis. The Specific Aims of the project are: 1) To examine the structure and function of the transport protein, utilizing FRET probes to look at cotransporter activation and conformational changes, testing a hypothesis of functionally interacting dimer partners, using cysteine scanning mutagenesis to investigate potential reentrant pore loops, and elucidating the mechanism of the activation switch in CCCs;2) To further elucidate the mechanism of regulation of NKCC1 by cell volume and intracellular [Cl-] utilizing a transgenic reporter mouse encoding a fluorescent sensor of intracellular Cl- concentration and transporter activation. Diseases and disease conditions including hypertension, cerebral edema, polycystic kidney disease, secretory diarrhea, cystic fibrosis, and some diseases of the nervous system involve defects or overactivity of the cellular machinery that is responsible for salt movements across cell membranes. This research is directed to understanding one part of that cellular machinery, a protein called the Na-K-Cl cotransporter (or NKCC) that is responsible for handling coordinated sodium, potassium and chloride movements. By understanding the molecular structure of the protein, the mechanics of its action, and the mechanism of its regulation we will be better able to design diagnostic and therapeutic agents and treat these disease states.